US9522217B2 - Medical device with coating for capturing genetically-altered cells and methods for using same - Google Patents
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- US9522217B2 US9522217B2 US10/835,767 US83576704A US9522217B2 US 9522217 B2 US9522217 B2 US 9522217B2 US 83576704 A US83576704 A US 83576704A US 9522217 B2 US9522217 B2 US 9522217B2
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- VJWBCKFXBCDSBZ-UHFFFAOYSA-N C.C=C(CCC(=O)O)OO.C=C(CCCCC(=O)Cl)Cl=O.O=S(Cl)Cl Chemical compound C.C=C(CCC(=O)O)OO.C=C(CCCCC(=O)Cl)Cl=O.O=S(Cl)Cl VJWBCKFXBCDSBZ-UHFFFAOYSA-N 0.000 description 1
- VSDZBYYWYGLTNJ-UHFFFAOYSA-N C1CCCCCCC1.C=C(O)CCCO.CN1CC2CCCCCCC2C1.CN1CC2CCCCCCC2C1.CN1CCCC1.[H]C(=O)C(=C=C)C1C2CCCCCCC2CN1C Chemical compound C1CCCCCCC1.C=C(O)CCCO.CN1CC2CCCCCCC2C1.CN1CC2CCCCCCC2C1.CN1CCCC1.[H]C(=O)C(=C=C)C1C2CCCCCCC2CN1C VSDZBYYWYGLTNJ-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/08—Carbon ; Graphite
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/30—Inorganic materials
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
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- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
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- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
- A61L29/16—Biologically active materials, e.g. therapeutic substances
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/082—Inorganic materials
- A61L31/084—Carbon; Graphite
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- A—HUMAN NECESSITIES
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/10—Macromolecular materials
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- A—HUMAN NECESSITIES
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/12—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/45—Mixtures of two or more drugs, e.g. synergistic mixtures
Definitions
- the invention relates to medical devices for implantation into vessels or hollowed organs of patients such as coated stents, stent grafts, synthetic vascular grafts, heart valves, catheters and vascular prosthetic filters for treating various diseases.
- the invention relates to medical devices comprising a coating on the surface that contacts blood, which coating is engineered to capture cells on the surface of the device.
- the captured cells form a monolayer on the surface of the device and are useful in many therapeutic applications, such as a drug delivery system or in the treatment of vascular disease.
- the cells binding to the implanted medical device may be native, progenitor endothelial cells from the circulating blood or genetically modified in vitro to express and secrete molecules or substances in vivo having a local or generalized therapeutic effect in the patient.
- Atherosclerosis involves the development of fatty plaques on the luminal surface of arteries. These fatty plaques causes narrowing of the cross-sectional area of the artery. Ultimately, blood flow distal to the lesion is reduced causing ischemic damage to the tissues supplied by the artery.
- Coronary arteries supply the heart with blood.
- Coronary artherosclerosis or coronary artery disease (CAD) is the most common, serious, chronic, life-threatening illness in the United States, affecting more than 11 million persons.
- the social and economic costs of coronary atherosclerosis vastly exceed those of most other diseases. Narrowing of the coronary artery lumen affects heart muscle resulting first in angina, followed by myocardial infarction and finally death, and more than three hundred thousand of those patients die before reaching the hospital. ( Harrison's Principles of Internal Medicine, 14th Edition, 1998).
- CAD can be treated using percutaneous translumenal coronary angioplasty (PTCA). More than 400,000 PTCA procedures are performed each year in the United States.
- PTCA percutaneous translumenal coronary angioplasty
- a balloon catheter is inserted into a peripheral artery and threaded through the arterial system into the blocked coronary artery.
- the balloon is then inflated, the artery stretched, and the obstructing fatty plaque flattened, thereby increasing the cross-sectional flow of blood through the affected artery.
- the therapy does not usually result in a permanent opening of the affected coronary artery. As many as 50% of the patients who are treated by PTCA require a repeat procedure within six months to correct a re-narrowing of the coronary artery.
- restenosis this re-narrowing of the artery after treatment by PTCA is called restenosis.
- Acutely, restenosis involves recoil and shrinkage of the vessel.
- recoil and shrinkage of the vessel are followed by proliferation of medial smooth muscle cells in response to injury of the artery from PTCA.
- proliferation of smooth muscle cells is mediated by release of various inflammatory factors from the injured area including thromboxane A 2 , platelet derived growth factor (PDGF) and fibroblast growth factor (FGF).
- PDGF platelet derived growth factor
- FGF fibroblast growth factor
- Stents are metal scaffolds that are positioned in the diseased vessel segment to create a normal vessel lumen. Placement of the stent in the affected arterial segment prevents recoil and subsequent closing of the artery. Stents can also prevent local dissection of the artery along the medial layer of the artery. By maintaining a larger lumen than that created using PTCA alone, stents reduce restenosis by as much as 30%. Despite their success, stents have not eliminated restenosis entirely. (Suryapranata et al. 1998. Randomized comparison of coronary stenting with balloon angioplasty in selected patients with acute myocardial infarction. Circulation 97:2502-2502).
- Narrowing of the arteries can occur in vessels other than the coronary arteries, including the aortoiliac, infrainguinal, distal profunda femoris, distal popliteal, tibial, subclavian and mesenteric arteries.
- PID peripheral artery atherosclerosis disease
- the prevalence of peripheral artery atherosclerosis disease (PAD) depends on the particular anatomic site affected as well as the criteria used for diagnosis of the occlusion.
- physicians have used the test of intermittent claudication to determine whether PAD is present. However, this measure may vastly underestimate the actual incidence of the disease in the population. Rates of PAD appear to vary with age, with an increasing incidence of PAD in older individuals.
- PAD can be treated using percutaneous translumenal balloon angioplasty (PTA).
- PTA percutaneous translumenal balloon angioplasty
- the use of stents in conjunction with PTA decreases the incidence of restenosis.
- the post-operative results obtained with medical devices such as stents do not match the results obtained using standard operative revascularization procedures, i.e., those using a venous or prosthetic bypass material.
- PAD is treated using bypass procedures where the blocked section of the artery is bypassed using a graft.
- the graft can consist of an autologous venous segment such as the saphenous vein or a synthetic graft such as one made of polyester, polytetrafluoroethylene (PTFE), or expanded polytetrafluoroethylene (ePTFE), or other polymeric materials.
- PTFE polytetrafluoroethylene
- ePTFE expanded polytetrafluoroethylene
- stents With stents, the approach has been to coat the stents with various anti-thrombotic or anti-restenotic agents in order to reduce thrombosis and restenosis.
- impregnating stents with radioactive material appears to inhibit restenosis by inhibiting migration and proliferation of myofibroblasts.
- Irradiation of the treated vessel can cause severe edge restenosis problems for the patient.
- irradiation does not permit uniform treatment of the affected vessel.
- stents have also been coated with chemical agents such as heparin, phosphorylcholine, rapamycin, and taxol, all of which appear to decrease thrombosis and/or restenosis.
- chemical agents such as heparin, phosphorylcholine, rapamycin, and taxol, all of which appear to decrease thrombosis and/or restenosis.
- heparin and phosphorylcholine appear to markedly reduce thrombosis in animal models in the short term, treatment with these agents appears to have no long-term effect on preventing restenosis. Additionally, heparin can induce thrombocytopenia, leading to severe thromboembolic complications such as stroke. Therefore, it is not feasible to load stents with sufficient therapeutically effective quantities of either heparin or phosphorylcholine to make treatment of restenosis in this manner practical.
- Synthetic grafts have been treated in a variety of ways to reduce postoperative restenosis and thrombosis. (Bos et al. 1998. Small-Diameter Vascular Graft Prostheses: Current Status Archives Physio. Biochem. 106:100-115). For example, composites of polyurethane such as meshed polycarbonate urethane have been reported to reduce restenosis as compared with ePTFE grafts. The surface of the graft has also been modified using radiofrequency glow discharge to fluorinate the polyterephthalate graft. Synthetic grafts have also been impregnated with biomolecules such as collagen. However, none of these approaches has significantly reduced the incidence of thrombosis or restenosis over an extended period of time.
- the endothelial cell (EC) layer is a crucial component of the normal vascular wall, providing an interface between the bloodstream and the surrounding tissue of the blood vessel wall. Endothelial cells are also involved in physiological events including angiogenesis, inflammation and the prevention of thrombosis (Rodgers G M. FASEB J 1988; 2:116-123.). In addition to the endothelial cells that compose the vasculature, recent studies have revealed that ECs and endothelial progenitor cells (EPCs) circulate postnatally in the peripheral blood (Asahara T, et al. Science 1997; 275:964-7; Yin A H, et al. Blood 1997; 90:5002-5012; Shi Q, et al.
- EPCs endothelial progenitor cells
- EPCs are believed to migrate to regions of the circulatory system with an injured endothelial lining, including sites of traumatic and ischemic injury (Takahashi T, et al. Nat Med 1999; 5:434-438). In normal adults, the concentration of EPCs in peripheral blood is 3-10 cells/mm 3 (Takahashi T, et al. Nat Med 1999; 5:434-438; Kalka C, et al. Ann Thorac Surg. 2000; 70:829-834).
- VEGF vascular endothelial growth factor
- Synthetic grafts have also been seeded with endothelial cells, but the clinical results with endothelial seeding have been generally poor, i.e., low post-operative patency rates (Lio et al. 1998. New concepts and Materials in Microvascular Grafting: Prosthetic Graft Endothelial Cell Seeding and Gene Therapy. Microsurgery 18:263-256) due most likely to the fact the cells did not adhere properly to the graft and/or lost their EC function due to ex-vivo manipulation.
- Endothelial cell growth factors and environmental conditions in situ are therefore essential in modulating endothelial cell adherence, growth and differentiation at the site of blood vessel injury. Accordingly, with respect to restenosis and other blood vessel diseases, there is a need for the development of new methods and compositions for coating medical devices, including stents and synthetic grafts, which would promote and accelerate the formation of a functional endothelium on the surface of implanted devices so that a confluent EC monolayer is formed on the target blood vessel segment or grafted lumen thereby inhibiting neo-intimal hyperplasia.
- liposomes to deliver drugs has been advantageous in that, in general, they increase the drug circulation time in blood, reduce side effects by limiting the concentration of free drug in the bloodstream, decrease drug degradation, prolong the therapeutic effect after each administration, reduce the need for frequent administration, and reduce the amount of drug needed.
- liposome systems that are currently available show limited efficiency of delivering drugs to target sites in vivo. See Kaye et al., 1979, Poznansky et al. 1984, U.S. Pat. Nos. 5,043,165, and 4,920,016.
- drug eluting stents More recently local drug delivery vehicles such as drug eluting stents (DES) have been developed. See U.S. Pat. Nos. 6,273,913, 6,258,121, and 6,231,600.
- drug eluting stents of the prior art are limited by many factors such as, the type of drug, the amount of drug to be released and the amount of time it takes to release the drug.
- Other factors which need to be considered in regards to drug eluting stents are the drug interactions with other stent coating components, such as polymer matrices, and individual drug properties including hydrophobicity, molecular weight, intactness and activity after sterilization, as well as efficacy and toxicity.
- polymer matrices of drug eluting stents one must consider the polymer type, polymer ratio, drug loading capability, and biocompatibility of the polymer and the drug-polymer compatibility such as drug pharmacokinetics.
- drug dose in a drug eluting stent is pre-loaded and an adjustment of drug dose upon individual conditions and need cannot be achieved.
- drug release time drug eluting stents instantly start to release the drug upon implantation and an ideal real-time release cannot be achieved.
- the present invention provides a system for the delivery of therapeutic agents locally or systemically in a safe and controlled manner.
- the therapeutic or drug delivery system comprises a medical device with a coating composed of a matrix comprising at least one type of ligand for recognizing and binding target cells such as progenitor endothelial cells or genetically-altered mammalian cells and genetically-altered mammalian cells which have been at least singly or dually-transfected.
- the medical device of the invention can be any device that is implantable into a patient.
- the device is for insertion into the lumen of a blood vessels or a hollowed organ, such as stents, stent grafts, heart valves, catheters, vascular prosthetic filters, artificial heart, external and internal left ventricular assist devices (LVADs), and synthetic vascular grafts, for the treatment of diseases such as cancer, vascular diseases, including, restenosis, artherosclerosis, thrombosis, blood vessel obstruction, or any other applications additionally covered by these devices.
- a blood vessels or a hollowed organ such as stents, stent grafts, heart valves, catheters, vascular prosthetic filters, artificial heart, external and internal left ventricular assist devices (LVADs), and synthetic vascular grafts, for the treatment of diseases such as cancer, vascular diseases, including, restenosis, artherosclerosis, thrombosis, blood vessel obstruction, or any other applications additionally covered by these devices.
- diseases
- the coating on the present medical device comprises a biocompatible matrix and at least one type of substance or ligand, which specifically recognize and bind target cells such as progenitor endothelial cells such as in the prevention or treatment of restenosis, or genetically-altered mammalian cells, onto the surface of the device, such as in the treatment of blood vessel remodeling and cancer.
- target cells such as progenitor endothelial cells such as in the prevention or treatment of restenosis, or genetically-altered mammalian cells
- the coating of the medical device may optionally comprise at least an activating compound for regulating the expression and secretion of the engineered genes of the genetically-altered cells.
- activator stimulatory compounds include but is not limited to chemical moieties, and peptides, such as growth factors.
- the coating when the coating comprises at least one compound, the activator molecule or compound may function to stimulate the cells to express and/or secrete at least one therapeutic substance for the treatment of disease.
- the coating on the medical device comprises a biocompatible matrix which comprises an outer surface for attaching a therapeutically effective amount of at least one type of ligand such as an antibody, antibody fragment, or a combination of the antibody and the antibody fragment, or at least one type of molecule for binding the engineered marker on the surface of the genetically-modified cell.
- the present antibody or antibody fragment recognizes and binds an antigen or the specific genetically-engineered cell surface marker on the cell membrane or surface of target cells so that the cells are immobilized on the surface of the device.
- the coating may optionally comprise an effective amount of at least one compound for stimulating the immobilized progenitor endothelial cells to either accelerate the formation of a mature, functional endothelium if the target cells are circulating progenitor cells, or to stimulate the bound cells to express and secrete the desired gene products if the target are genetically-altered cells on the surface of the medical device.
- the medical device of the invention can be any device used for implanting into an organ or body part comprising a lumen, and can be, but is not limited to, a stent, a stent graft, a synthetic vascular graft, a heart valve, a catheter, a vascular prosthetic filter, a pacemaker, a pacemaker lead, a defibrillator, a patent foramen ovale (PFO) septal closure device, a vascular clip, a vascular aneurysm occluder, a hemodialysis graft, a hemodialysis catheter, an atrioventricular shunt, an aortic aneurysm graft device or components, a venous valve, a suture, a vascular anastomosis clip, an indwelling venous or arterial catheter, a vascular sheath and a drug delivery port.
- a stent a stent graft, a synthetic vascular graft
- the medical device can be made of numerous materials depending on the device.
- a stent of the invention can be made of stainless steel, Nitinol (NiTi), or chromium alloy and biodegradable materials.
- Synthetic vascular grafts can be made of a cross-linked PVA hydrogel, polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), porous high density polyethylene (HDPE), polyurethane, and polyethylene terephthalate, or biodegradable materials.
- the biocompatible matrix forming the coating of the present medical device comprises without limitation a synthetic material such as polyurethanes, segmented polyurethane-urea/heparin, poly-L-lactic acid, cellulose ester, polyethylene glycol, polyvinyl acetate, dextran and gelatin, and/or naturally-occurring material such as basement membrane components such as collagen, elastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose, and amorphous carbon, or fullerenes.
- a synthetic material such as polyurethanes, segmented polyurethane-urea/heparin, poly-L-lactic acid, cellulose ester, polyethylene glycol, polyvinyl acetate, dextran and gelatin, and/or naturally-occurring material such as basement membrane components such as collagen, elastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose, and amorphous carbon, or
- the medical device comprises a biocompatible matrix comprising fullerenes.
- the fullerene can range from about C 20 to about C 150 in the number of carbon atoms, and more particularly, the fullerene is C 60 or C 70 .
- the fullerene of the invention can also be arranged as nanotubes on the surface of the medical device.
- the ligand is applied to the blood contacting surface of the medical device and the ligand specifically recognizes and binds a desired component or epitope on the surface of target cells in the circulating blood.
- the ligand is specifically designed to recognize and bind only the genetically-altered mammalian cell by recognizing only the genetically-engineered marker molecule on the cell membrane of the genetically-altered cells. The binding of the target cells immobilizes the cells on the surface of the device.
- the antibody can be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a humanized antibody which recognizes and binds only to the genetically-altered endothelial cell by interacting with the surface marker molecule and, thereby modulating the adherence of the cells onto the surface of the medical device.
- the antibody or antibody fragment of the invention can be covalently or noncovalently attached to the surface of the matrix, or tethered covalently by a linker molecule to the outermost layer of the matrix coating the medical device.
- the monoclonal antibodies can further comprises Fab or F(ab′) 2 fragments.
- the antibody fragment of the invention comprises any fragment size, such as large and small molecules which retain the characteristic to recognize and bind the target antigen as the antibody.
- the antibody or antibody fragment of the invention recognize and bind antigens with specificity for the mammal being treated and their specificity is not dependent on cell lineage.
- the antibody or fragment is specific for selecting and binding circulating progenitor endothelial cell surface antigen such as CD133, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146), CD130, stem cell antigen (Sca-1), stem cell factor 1 (SCF/c-Kit ligand), Tie-2 and HAD-DR.
- the coating of the medical device comprises at least one layer of a biocompatible matrix as described above, the matrix comprising an outer surface for attaching a therapeutically effective amount of at least one type of small molecule of natural or synthetic origin.
- the small molecule recognizes and interacts with, for example, progenitor endothelial cells in the treatment of restenosis, to immobilize the cells on the surface of the device to form an endothelial layer.
- the small molecules can be used in conjunction with the medical device for the treatment of various diseases, and can be derived from a variety of sources such as cellular components such as fatty acids, proteins, nucleic acids, saccharides and the like and can interact with an antigen on the surface of a progenitor endothelial cell with the same results or effects as an antibody.
- the coating on the medical device can further comprise a compound such as a growth factor as described herewith in conjunction with the coating comprising an antibody or antibody fragment.
- the compound of the coating of the invention for example in treating restenosis, comprises any compound which stimulates or accelerates the growth and differentiation of the progenitor cell into mature, functional endothelial cells.
- the compound is for stimulating the genetically modified cells to express and secrete the desired gene product.
- the activating agents or compounds useful for stimulating the cells to express and secrete the genetically-engineered gene products include, but are not limited to estrogen, tetracycline and other antibiotics, tamoxiphen, etc., and can be provided to the patient via various routes of administration, such as through the skin via a patch and subcutaneously.
- the invention also provides methods for treating a variety of diseases, such as vascular disease, cancer, blood vessel remodeling, severe coronary artery disease. artherosclerosis, restenosis, thrombosis, aneurysm and blood vessel obstruction.
- the method provides an improvement over prior art methods as far as retaining or sealing the medical device insert to the vessel wall, such as a stent or synthetic vascular graft, heart valve, abdominal aortic aneurysm devices and components thereof, for establishing vascular homeostasis, and thereby preventing excessive intimal hyperplasia as in restenosis.
- the artery may be either a coronary artery or a peripheral artery such as the femoral artery.
- Veins can also be treated using the techniques and medical device of the invention.
- the genetically-altered cells are provided with exogenous genetic material to introduce at least one desired gene which encodes a cell surface marker and at least one gene which encodes a therapeutic gene product.
- the system optionally comprises a signal system, such as an activating compound or molecule for stimulating the genetically-altered mammalian cells to express and/or secrete the desired gene product and/or the marker gene.
- the medical device of the invention comprises a coating for the specific in vivo capturing and immobilization of genetically-altered mammalian cells which are introduced, simultaneously or sequentially, into the patient upon implantation of the coated medical device.
- the cells e.g., endothelial cells are genetically-altered by introducing exogenous genetic material into the cells.
- the genetic material is introduced into the nucleus of the cells and is DNA, such as extrachromosomal DNA.
- the extrachromosomal DNA may be a vector such an adenoviral vector, a plasmid such as a naked plasmid, linear or short DNA, and the like.
- the DNA comprises regulatory/expression cassette for controlling the expression of the desired marker and/or therapeutic genes.
- the regulatory cassette may comprise regulatory elements for constitutive expression of the therapeutic genes or may comprise elements that can be controlled or expressed as needed by the patient.
- the medical device for implantation into the patient comprises a coating; the coating comprises a matrix bearing at least one type of ligand, which recognizes and binds target cells.
- the ligand only recognizes a specific cell membrane marker which is engineered into the cells.
- such ligand only recognizes the genetically-altered mammalian cells introduced into the patient, and the genetically-altered mammalian cells bind to said medical device and express and secrete said at least one therapeutic gene product.
- the therapeutic or drug delivery system may further comprise an activating molecule for stimulating said genetically-altered mammalian cells to express and/or secrete the desired therapeutic gene products.
- a compound such as a chemical stimulus or a peptide can be provided to the patient via an oral route, a thermal patch, intravenously, intradermally and the like.
- the genetically-altered mammalian cells may be autogenic or xenogenic, such as mature endothelial cells, fibroblasts, muscle cells, epithelial cells, etc. exogenous nucleic acid is extrachromosomal DNA.
- the DNA is provided in the form of a vector, such as an adenovirus vector, naked plasmid DNA, linear DNA and the like.
- the extrachromosomal DNA comprises a regulatory cassette, a gene which encodes a cell membrane antigen and at least one gene which encodes a peptide for treating a disease.
- the cell membrane specific gene encodes, for example, an osteogenic or a prostatic cell membrane protein.
- genetically-altered mammalian cells comprising exogenous nucleic acid encoding a genetically-engineered cell membrane marker and at least one therapeutic gene product;
- the invention also provides a method for treating disease in a patient, the method comprises: providing genetically-altered mammalian cells to the patient; implanting a medical device into the patient; wherein the medical device comprises a coating which comprises a matrix bearing at least one ligand, wherein the ligand specifically recognizes and binds at least one receptor on the genetically-altered mammalian cells, and wherein the genetically-altered mammalian cells bind to the medical device and comprise exogenous nucleic acid for expressing and secreting a therapeutic gene product.
- the blood contacting surface can be a biodegradable scaffolding or can be coated with a biodegradable, biocompatible material.
- the biodegradable scaffolding when implanted into a blood vessel undergoes in situ degradation and the neo-endothelium formed on the luminal surface of the device restores the blood vessel continuity through the injured site so as to form a functional neo-vessel.
- the invention comprises prosthesis, comprising: (a) a support member having an exterior surface and a blood contacting surface; (b) a first layer of a cross-linked polymeric compound coated onto said blood contacting surface of said support member; and, (c) a second layer coated on said first layer, said second layer comprising at least one ligand having an affinity for a targeted cell in vivo.
- a method for generating a self-endothelializing graft in vivo comprising: (a) providing a scaffolding configured to function as a vascular graft, said scaffolding having a lumen surface and exterior surface, said lumen surface comprising ligands specific for binding to endothelial progenitor cells; (b) implanting said scaffolding into a blood vessel of a subject; and (c) recruiting circulating endothelial progenitor cells to said lumen surface of said scaffolding to form a neo-endothelium.
- a method for generating a self-endothelializing graft in situ comprising: (a) providing a prosthetic structure having a surface exposed to circulating blood; (b) implanting the prosthetic structure into a subject; and (c) recruiting circulating endothelial progenitor cells from the blood to the surface of the prosthetic structure to form a neo-endothelium thereon.
- FIG. 1A is a schematic representation of an antibody tethered covalently to the matrix by a cross-linking molecule.
- FIG. 1B shows a diagram of the C 60 O molecule anchoring the matrix.
- FIG. 1C depicts a schematic representation of a stent coated with the matrix of the invention showing how cells interact with ligands on the surface of a device.
- FIG. 2A is a phase contrast micrograph of progenitor endothelial cells adhered to a fibronectin-coated slide containing cells isolated by enriched medium.
- FIG. 2B is a phase contrast micrograph of progenitor endothelial cells adhered to a fibronectin-coated slide containing cells isolated by anti-CD34 antibody coated magnetic beads.
- FIGS. 2D and 2F are micrographs of the progenitor endothelial cells which had been incubated for 7 days and stained with PI nuclear stain. As seen in these figures, the cells express mature endothelial cell markers as shown by the antibody fluorescence for Tie-2 ( FIGS. 2E and 2G ) and VEGFR-2 ( FIG. 2C ) antibody reactivity.
- FIGS. 6A-6C are photomicrographs of stainless steel discs coated with CMDx matrix with anti-CD34 antibody bound to its surface which were incubated with the HUVECs. The cells were stained with propidium iodide and FITC labeled anti-KDR antibody.
- FIGS. 6D-6F are photomicrographs of stainless steel discs coated with gelatin matrix with antibody bound to its surface, which were incubated with HUVECS. The cells were stained with propidium iodide and FITC labeled anti-KDR antibody.
- FIGS. 8A and 8B are photomicrographs of a stainless steel disc coated with CMDx matrix containing anti-CD34 antibody bound to its surface incubated with progenitor cells for 7 days. The cells were stained with propidium iodide and FITC labeled anti-KDR antibody.
- FIGS. 10A-10C are phase contrast photomicrographs of stainless steel CMDx coated discs incubated with progenitor cells for 3 weeks in endothelial growth medium which show mature endothelial cells.
- FIG. 11 is schematic diagram of a functional fullerene coated stent surface of the invention binding a progenitor cell.
- FIGS. 14A-14G are scanning electron micrographs of stent explants 1 and 48 hours after implantation in male Yorkshire swine. Explants of dextran-coated ( FIG. 14A ) and dextran/anti-CD34 antibody-coated ( 14 B) stents at 1 hour after implantation. FIGS. 14C and 14D show explants of control samples and FIGS. 14E-G are dextran/anti-CD34 antibody-coated stents at 48 hours after implantation. FIGS.
- the matrix comprises a synthetic or naturally-occurring material in which a therapeutically effective amount of at least one type of antibody that promotes adherence of endothelial, progenitor or stem cells to the medical device, and at least one compound such as a growth factor, which stimulates endothelial cell growth and differentiation.
- the cells that adhere to the surface of the device transform into a mature, confluent, functional layer of endothelium on the luminal surface of the medical device.
- the presence of a confluent layer of endothelial cells on the medical device reduces the occurrence of restenosis and thrombosis at the site of implantation.
- Coating of the medical device with the compositions and methods of this invention stimulates the development of a confluent mammalian cell layer such as an endothelial cell layer on the surface of the medical device, thereby preventing restenosis as well as modulating the local chronic inflammatory response and thromboembolic complications that result from implantation of the medical device.
- a confluent mammalian cell layer such as an endothelial cell layer
- the matrix coating the medical device can be composed of synthetic material, such as polymeric gel foams, such as hydrogels made from polyvinyl alcohol (PVA), polyurethane, poly-L-lactic acid, cellulose ester or polyethylene glycol.
- synthetic material such as polymeric gel foams, such as hydrogels made from polyvinyl alcohol (PVA), polyurethane, poly-L-lactic acid, cellulose ester or polyethylene glycol.
- PVA polyvinyl alcohol
- very hydrophilic compounds such as dextran compounds can comprise the synthetic material for making the matrix.
- the matrix is composed of naturally occurring materials, such as collagen, fibrin, elastin, or amorphous carbon.
- the matrix may comprise several layers with a first layer being composed of synthetic or naturally occurring materials and a second layer composed of antibodies. The layers may be ordered sequentially, with the first layer directly in contact with the stent or synthetic graft surface and the second layer having one surface in contact with the first layer and the opposite surface in contact with the vessel lumen.
- the term “therapeutically effective amounts of growth factor” means the amount of a growth factor that stimulates or induces endothelial, progenitor or stem cells to grow and differentiate, thereby forming a confluent layer of mature and functional endothelial cells on the luminal surface of the medical device.
- the amount of a growth factor needed to practice the invention varies with the nature of the growth factor used and binding kinetics between the growth factor and its receptor. For example, 100 ⁇ g of VEGF has been shown to stimulate the adherence of endothelial cells on a medical device and form a confluent layer of epithelium. It is well known to those of ordinary skill in the art how to determine therapeutically effective amounts of a growth factor to use to stimulate cell growth and differentiation of endothelial cells.
- the subjects that can be treated using the medical device, methods and compositions of this invention are mammals, or more specifically, a human, dog, cat, pig, rodent or monkey.
- porcine progenitor endothelial cells are transfected with vascular endothelial growth factor (VEGF) using an adenoviral expression vector expressing the VEGF cDNA according to the methods of Rosengart et al. (Six-month assessment of a phase I trial of angiogenic gene therapy for the treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF121 cDNA. Ann. Surg. 230(4):466-470, 1999, incorporated herein by reference).
- the mammalian cells can be autologous, allogenic or xenogenic in origin.
- the stent can be composed of polymeric or metallic structural elements onto which the matrix comprising the antibodies and the compound, such as growth factors, is applied or the stent can be a composite of the matrix intermixed with a polymer.
- a deformable metal wire stent can be used, such as that disclosed in U.S. Pat. No. 4,886,062 to Wiktor, incorporated herein by reference.
- a self-expanding stent of resilient polymeric material such as that disclosed in published international patent application WO91/12779 “Intraluminal Drug Eluting Prosthesis”, incorporated herein by reference, can also be used.
- any biocompatible synthetic graft can be used with the antibodies, growth factors, and matrices of this invention.
- Synthetic grafts can be used for end-to-end, end to side, side to end, side to side or intraluminal and in anastomosis of vessels or for bypass of a diseased vessel segments, for example, as abdominal aortic aneurysm devices.
- the matrix may be selected from naturally occurring substances such as collagen, fibronectin, vitronectin, elastin, laminin, heparin, fibrin, cellulose or carbon.
- a primary requirement for the matrix is that it be sufficiently elastic and flexible to remain unruptured on the exposed surfaces of the stent or synthetic graft.
- the matrix may also comprise a fullerene (the term “fullerene” encompasses a plurality of fullerene molecules).
- Fullerenes are carbon-cage molecules. The number of carbon (C) molecules in a fullerene species varies from about C 20 to about C 150 .
- Fullerenes are produced by high temperature reactions of elemental carbon or of carbon-containing species by processes well known to those skilled in the art; for example, by laser vaporization of carbon, heating carbon in an electric arc or burning of hydrocarbons in sooting flames. (U.S. Pat. No. 5,292,813, to Patel et al., incorporated herein by reference; U.S. Pat. No.
- Fullerenes may be deposited on surfaces in a variety of different ways, including, sublimation, laser vaporization, sputtering, ion beam, spray coating, dip coating, roll-on brush coating as disclosed in U.S. Pat. No. 5,558,903, or by derivatization of the surface of the stent.
- lectins or antibodies can be adsorbed to the fullerene surface.
- Attachment of different molecules to the fullerene surface may be manipulated to create surfaces that selectively bind various cell types, e.g., progenitor endothelial cells, epithelial cells, fibroblasts, primary explants, or T-cell subpopulations.
- nanotubes may be filled with the enzymes, e.g., Zn 2 Cd 2 -metallothionein, cytochromes C and C3, and beta-lactamase after cutting the ends of the nanotube.
- enzymes e.g., Zn 2 Cd 2 -metallothionein, cytochromes C and C3, and beta-lactamase after cutting the ends of the nanotube.
- U.S. Pat. No. 5,338,571 to Mirkin et al. discloses three-dimensional, multilayer fullerene structures that are formed on a substrate surface by (i) chemically modifying fullerenes to provide a bond-forming species; (ii) chemically treating a surface of the substrate to provide a bond-forming species effective to covalently bond with the bond-forming species of the fullerenes in solution; and, (iii) contacting a solution of modified fullerenes with the treated substrate surface to form a fullerene layer covalently bonded to the treated substrate surface.
- the stent surface is first functionalized, followed by the addition of a matrix layer. Thereafter, the antibodies and the growth factor are coupled to the surface of the matrix.
- the techniques of the stent surface creates chemical groups which are functional. The chemical groups such as amines, are then used to immobilize an intermediate layer of matrix, which serves as support for the antibodies and the growth factor.
- a suitable matrix coating solution is prepared by dissolving 480 milligrams (mg) of a drug carrier, such as poly-D, L-lactid (available as R203 of Boehringer Inc., Ingelheim, Germany) in 3 milliliters (ml) of chloroform under aseptic conditions.
- a drug carrier such as poly-D, L-lactid (available as R203 of Boehringer Inc., Ingelheim, Germany) in 3 milliliters (ml) of chloroform under aseptic conditions.
- any biodegradable (or non-biodegradable) matrix that is blood- and tissue-compatible (biocompatible) and can be dissolved, dispersed or emulsified may be used as the matrix if, after application, it undergoes relatively rapid drying to a self-adhesive lacquer- or paint-like coating on the medical device.
- the dispersion is typically injected via syringe into the lumen of a graft and massaged manually to cover the entire inner surface area with the collagen slurry. Excess collagen slurry is removed through one of the open ends of the graft. Coating and drying steps are repeated several times to provide sufficient treatment.
- Fullerenes may also be attached through an epoxide bond to the surface of stainless steel (Yamago et al., Chemical Derivatization of Organofullerenes through Oxidation, Reduction and C—O and C—C Bond Forming Reactions. J. Org. Chem., 58 4796-4798 (1998), incorporated herein by reference).
- the attachment is through a covalent linkage to the oxygen.
- This compound and the protocols for coupling are commercially available from BuckyUSA. (BuckyUSA, Houston, Tex.).
- Antibodies that promote adherence of progenitor endothelial cells, and growth factors for promoting cell growth and differentiation are incorporated into the matrix, either covalently or noncovalently.
- Antibodies and growth factor may be incorporated into the matrix layer by mixing the antibodies and growth factor with the matrix coating solution and then applied to the surface of the device.
- antibodies and growth factors are attached to the surface of the outermost layer of matrix that is applied on the luminal surface of the device, so that the antibodies and growth factor are projecting on the surface that is in contact with the circulating blood.
- Antibodies and growth factors are applied to the surface matrix using standard techniques.
- the resulting polymerized fibrin mixture containing the Fab fragments incorporated directly into the matrix is pressed into a thin film (less than 100 ⁇ m) on the surface of the stent or synthetic graft.
- a thin film (less than 100 ⁇ m) on the surface of the stent or synthetic graft.
- Virtually any type of antibody or antibody fragment can be incorporated in this manner into a matrix solution prior to coating of a stent or synthetic graft.
- Antibodies may be attached to C 60 fullerene layers that have been deposited directly on the surface of the stent.
- Cross linking agents may be covalently attached to the fullerenes.
- the antibodies are then attached to the cross-linking agent, which in turn is attached to the stent.
- FIG. 1B provides an illustration of coupling by C 60 .
- the endothelial cell, 2.01 is bound via a cell surface antigen, 2.02, to an antibody, 2.03, which in turn is bound, covalently or non-covalently to the matrix, 2.04.
- the matrix, 2.04, is coupled covalently via C 60 , 2.05, to the stent, 2.06.
- Small molecules of the invention comprise synthetic or naturally occurring molecules or peptides which can be used in place of antibodies, growth factors or fragments thereof.
- lectin is a sugar-binding peptide of non-immune origin which occurs naturally.
- the endothelial cell specific Lectin antigen (Ulex Europaeus Uea 1) (Schatz et al. 2000 Human Endometrial Endothelial Cells: Isolation, Characterization, and Inflammatory-Mediated Expression of Tissue Factor and Type 1 Plasminogen Activator Inhibitor. Biol Reprod 62: 691-697) can selectively bind the cell surface of progenitor endothelial cells.
- Synthetic “small molecules” have been created to target various cell surface, proteins, glucoproteins, polysaccharides and receptors. These molecules selectively bind a specific surface moieties and can target specific cell types such as progenitor endothelial cells. Small molecules can be synthesized to recognize endothelial cell surface markers such as VEGF. SU11248 (Sugen Inc.) (Mendel et al. 2003 In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res .
- alpha(v)beta(3) integrin inhibitors Another subset of synthetic small molecules which target the endothelial cell surface are the alpha(v)beta(3) integrin inhibitors.
- SM256 and SD983 (Kerr J S. et al. 1999 Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.
- Anticancer Res March-April; 19(2A):959-68) are both synthetic molecules which target and bind to alpha(v)beta(3) present on the surface of endothelial cells.
- the present invention provides a drug delivery system comprising: coated medical devices such as stents, stent grafts, heart valves, catheters, vascular prosthetic filters, artificial heart, external and internal left ventricular assist devices (LVADs), and synthetic vascular grafts, for the treatment of diseases, including tumor and vascular diseases, such as restenosis, artherosclerosis, thrombosis, blood vessel obstruction, and the like.
- coated medical devices such as stents, stent grafts, heart valves, catheters, vascular prosthetic filters, artificial heart, external and internal left ventricular assist devices (LVADs), and synthetic vascular grafts, for the treatment of diseases, including tumor and vascular diseases, such as restenosis, artherosclerosis, thrombosis, blood vessel obstruction, and the like.
- the coating on the present medical device comprises a biocompatible matrix, at least one antibody, and at least one compound such as a ligand.
- Transgenic cells incorporating at least one transgene that is introduced into the cells by viral or non-viral based genetic procedures.
- the transgene codes for at least one therapeutic drug and is expressed continuously or upon induction.
- the therapeutic drug is a protein.
- the transgenic cells also present at least one antigen on its cell surface that can be recognized and bound by the antibody that is coated on the surface of the medical device.
- antibody refers to antibody or antibody fragment, or a combination of antibody and fragments, which can be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a humanized antibody.
- the antibody fragment of the invention comprises any fragment size, such as large and small molecules which retain the characteristic to recognize and bind the target antigen as the antibody ( FIGS. 1A, 1B, and 11 ).
- ligand refers to a molecule that binds a receptor on the mammalian cell.
- the ligand can be an antibody, antibody fragment ( FIGS. 1A, 1B, 11, and 17 ), cell adhesion molecule, basement membrane component which recognizes and binds a specific epitope or structure on the membrane of the cell.
- the ligand can be specifically selected to recognize and bind to a gene product produced by the exogenous DNA introduced into the cells.
- the medical device of this invention can be any device used for implanting into an organ or body part comprising a lumen, and can be, but is not limited to, a stent, a stent graft, a synthetic vascular graft, a heart valve, a catheter, a vascular prosthetic filter, a pacemaker, a pacemaker lead, a defibrillator, a patent foramen ovale (PFO) septal closure device, a vascular clip, a vascular aneurysm occluder, a hemodialysis graft, a hemodialysis catheter, an atrioventricular shunt, an aortic aneurysm graft device or components, a venous valve, a suture, a vascular anastomosis clip, an indwelling venous or arterial catheter, a vascular sheath and a drug delivery port.
- a stent a stent graft, a synthetic vascular graft
- the medical device comprises a biocompatible matrix comprising fullerenes.
- the fullerene can range from about C 20 to about C 150 in the number of carbon atoms, and more particularly, the fullerene is C 60 or C 70 .
- the fullerene of the invention can also be arranged as nanotubes on the surface of the medical device.
- the antibody of the invention recognizes and binds antigens with specificity for the mammal being treated and their specificity is not dependent on cell lineage.
- the antibody is specific for a human progenitor endothelial cell surface antigen such as CD133, CD14, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146), CD130, stem cell antigen (Sca-1), stem cell factor 1 (SCF/c-Kit ligand), Tie-2, HAD-DR and others.
- the coating of the medical device comprises at least one layer of a biocompatible matrix as described above, the matrix comprising an outer surface for attaching a therapeutically effective amount of at least one type of small molecule of natural or synthetic origin.
- the small molecule recognizes and interacts with an antigen on a transgenic cell surface to immobilize the transgenic cell on the surface of the device and to induce transgene expression.
- the small molecules can be derived from a variety of sources such as cellular components such as fatty acids, proteins, nucleic acids, saccharides and the like and can interact with a receptor on the surface of a transgenic cell.
- the coating on the medical device can further comprise a compound such as a ligand in conjunction with the coating comprising an antibody.
- Transgenic cells of the invention express and secrete therapeutic drugs coded by transgenes that are either transiently or stably incorporated. Additional transgenes can be incorporated to confer survival, selection and/or growth advantage.
- Various cells such as endothelial cells or leukocytes including neutrophil, eosinophil, basophil, monocyte and lymphocytes or somatic cells, or a combination of these cells can be modified to produce transgenic cells, which may be either non-repopulating or repopulating.
- Transgenic cells can be cultured in vitro, collected, and stored.
- Transgenic cells producing a variety of therapeutic drugs can be generated by incorporating different transgenes to serve different therapeutic purposes.
- Transgenic cells can be administered as a single or mixed populations via systemic or local routes. Various amounts of transgenic cells can be administered to release different amount of therapeutic drugs upon individual conditions.
- transgenic cells are repopulating progenitor endothelial cells.
- transgenic progenitor endothelial cells are administered locally with catheter based delivery or dual balloon inflation method.
- transgenic cells further comprise an additional transgene that expresses an exogenous cell surface antigen, which can be specifically recognized and bound by the antibody that is coated in the matrix of the medical device.
- Transgene expression and product secretion can be continuous or contingent upon the activation of an inducible promoter via exogenous excitation.
- the therapeutic compounds coded by the transgenes of the invention can be any molecule with a desired physiological effect, and can be, but is not limited to, proteins as defined including growth factors, chemokines and cytokines, ligands and receptors, and other functional proteins and non-protein excretable compounds.
- a therapeutic compound is a protein selected from the group consisting of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), an anti-inflammatory factor, and an inflammation-modulating factor.
- the exciting drug for transgene expression and product secretion of the invention can be a ligand that binds the transgenic cell surface antigen and triggers downstream signaling pathway activation, or can be taken up by the transgenic cell and stimulate gene expression through an inducible promoter.
- the ligand or drug is administered systemically.
- the ligand or drug is coated in the matrix of the implanted device and administered locally.
- the invention provides methods for treating a variety of diseases, which can be, but not limited to, tumors, vascular diseases, and healing response.
- the methods provide improvement over prior art in terms of target site delivery of a variety of drugs of desired amount upon demand.
- the transgene can code for (1) an antiangiogenic factor, such as interferons (IFNs), thrombospondin (TSP), angiostatin, endostatin, oncostatin M (OSM), and Rho, which inhibits neovascularization that is a prerequisite for tumor progressive growth; or (2) an tumor suppressive protein, such as p53, Rb, E1, BRCA1, antibody or dominant negative mutant of a cell growth activator such as a growth factor, a cyclin dependent kinase (CDK) or a cyclin, E2F, NF ⁇ B; or a combination of these genes.
- an antiangiogenic factor such as interferons (IFNs), thrombospondin (TSP), angiostatin, endostatin, oncostatin M (OSM), and Rho
- an antiangiogenic factor such as interferons (IFNs), thrombospondin (TSP), angiostatin, endostatin, oncostatin M (
- anti-angiogenic factor refers to a molecule that is capable of inhibiting angiogenesis.
- angiogenic factor refers to a molecule that is capable of stimulating angiogenesis.
- the transgenic (genetically-altered) cells contain genetic material which encodes at least one therapeutic drug constitutively or upon activation by a signal such as a compound.
- the present transgenic cells contain at least one expressible transgene that can code for, but not limited to (1) growth factors including family members such as platelet derived growth factor (PDGF), transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin like growth factors (IGF), vascular endothelial growth factor (VEGF), heparin binding growth factors, hepatoma-derived growth factor (HDGF), hepatocyte growth factor/scatter factor (HGF), placental growth factor (PIGF), platelet derived endothelial cell growth factor (PD-ECGF), stem cell factor (SCF), and their other protein forms; (2) Chemokines such as CXC families, CC families, C families, and their other protein forms; (3) cytokines such as a disintegrin and metalloprotease (ADAM), annexin V, B7 & CD28/CTLA-4 receptor families, bone morphogenetic protein (BMP), caspase, CD44, CD44H, endothel
- antiangiogenic factors for use in the invention are, for example, interferons (IFNs), thrombospondin (TSP), angiostatin, and endostatin, oncostatin M (OSM), blockers of integrin engagement, metalloproteinases inhibitors, inhibitors of endothelial cell phosphorylation, dominant negative receptors for angiogenesis inducers, antibodies of angiogenesis inducers, other proteins acting by other means, and their other protein forms.
- IFNs interferons
- TSP thrombospondin
- OSM oncostatin M
- blockers of integrin engagement metalloproteinases inhibitors
- inhibitors of endothelial cell phosphorylation inhibitors of endothelial cell phosphorylation
- dominant negative receptors for angiogenesis inducers antibodies of angiogenesis inducers
- Other angiogenic factors include angiogenin, angiopoietins, integrin stimulating factors such as Del-1, and their other protein forms.
- Additional growth factors for use in the invention are, for example, pleiotrophin, midkines, VEGF family including VEGF-2, VEGF-C, and VEGF-D, FGF family, including FGF-1, FGF-2, FGF-5, and FGF-18, hepatoma-derived growth factor (HDGF), hepatocyte growth factor/scatter factor (HGF), members of the epidermal growth factor (EGF) family, including transforming growth factor alpha, EGF, and TGF-alpha-HIII, and platelet derived growth factor (PDGF), including AA, AB, and BB isoforms.
- VEGF family including VEGF-2, VEGF-C, and VEGF-D
- FGF family including FGF-1, FGF-2, FGF-5, and FGF-18
- hepatoma-derived growth factor (HDGF) hepatocyte growth factor/scatter factor
- EGF epidermal growth factor
- PDGF platelet derived growth factor
- FIGS. 2C-2G The results of these experiments are shown in FIGS. 2C-2G .
- FIG. 2C shows that VEGFR-2 is expressed after 24 hours in culture, confirming that the cells are endothelial cells.
- FIGS. 2D and 2F show the nuclear staining of the bound cells after 7 days of incubation and FIGS. 2E and 2G the same field of cells stained with an FITC conjugated anti-Tie-2 antibody.
- HAVEC Human Umbilical Vein Endothelial Cells
- SST bare stainless steel
- PFA paraformaldehyde
- the samples are washed once with PBS and the exposed to 1:100 dilution of VEGFR-2 antibody and incubated overnight. The samples are subsequently washed three times with PBS to ensure all primary antibody has been removed.
- FITC-conjugated secondary antibody in blocking solution is added to each respective sample at a dilution of 1:100 and incubated for 45 minutes at room temperature on a Belly Dancer apparatus. After incubation, the samples are washed three times in PBS, once with PBS containing 0.1% Tween 20, and then again in PBS. The samples are mounted with Propidium Iodine (PI) and visualized under confocal microscopy.
- PI Propidium Iodine
- Progenitor cell are isolated from human blood as described in the in Example 1 and incubated in growth medium for 24 hours, 7 days, and 3 weeks in vitro. After incubation, the growth medium is removed and the samples are washed twice in PBS. Cells are fixed in 2% paraformaldehyde (PFA) for 10 minutes and washed three times, 10 minutes each wash, in PBS, to ensure all the fixing agent is removed. Each sample is incubated with 440 ⁇ l of Goat (for VEGFR-2) or Horse (for Tie-2) blocking solution for 30 minutes at room temperature, to block all non-specific binding.
- Goat for VEGFR-2
- Horse for Tie-2
- the samples are washed once with PBS and the VEGFR-2 or Tie-2 antibody was added at a dilution of 1:100 in blocking solution and the samples are incubated overnight. The samples are then washed three times with PBS to ensure all primary antibody has been washed away.
- FITC-conjugated secondary antibody 200 ⁇ l in horse or goat blocking solution is added to each respective sample at a dilution of 1:100 and incubated for 45 minutes at room temperature on a Belly Dancer apparatus. After incubation, the samples are washed three times in PBS, once with PBS containing 0.1% Tween 20, and then again in PBS. The samples are mounted with Propidium Iodine (PI) and visualized under confocal microscopy.
- PI Propidium Iodine
- FIG. 7 is a photomicrograph of a CMDX-coated sample containing CD34 antibody on its surface which was incubated with the cells for 24 hours, and shows that progenitor cells were captured on the surface of the sample and as demonstrated by the red-stained nuclei present on the surface of the sample. The figure also shows that about 75% of the cells are VEGFR-2 positive with a round morphology.
- FIGS. 8A and 8B are from a sample which was incubated with the cells for 7 days.
- FIG. 8A there are cells present on the sample as shown by the red-stained nuclei, which are VEGFR-2 positive ( FIG. 8B , 100%) and are more endothelial in structure as shown by the spindle shape of the cells.
- FIGS. 9A and 9B are photomicrographs of CMDX-coated sample containing CD34 antibody on its surface, which was incubated for 7 days with the cells and after incubation, the sample was exposed to Tie-2 antibody.
- FIG. 9A there are numerous cells attached to the surface of the samples as shown by the red-stained nuclei.
- the cells adhered to the sample are also Tie-2 positive (100%) as seen by the green fluorescence emitted from the cells ( FIG. 9B ).
- the CD34 antibody-coated samples are able to capture endothelial cells on their surface as seen by the numerous cells attached to the surface of the samples and the presence of VEGFR-2 and Tie-2 receptors on the surface of the adhered cells.
- the presence of 100% endothelial cells on the surface of the samples at 7 days indicates that the non-endothelial cells may have detached or that all adherent cells have begun to express endothelial cell markers by day 7.
- FIGS. 10A-10C are phase contrast photomicrographs of the progenitor endothelial cells grown for 3 weeks in endothelial cell growth medium.
- FIG. 10A demonstrates the cells have differentiated into matured endothelial cells as shown by the two-dimensional tube-like structures (arrow) reminiscent of a lumen of a blood vessel at the arrow.
- FIG. 10B shows that there is a three-dimensional build-up of cells in multiple layers; i.e.; one on top of the other, which confirms reports that endothelial cells grown for prolonged periods of time begin to form layers one on top of the other.
- FIG. 10A shows the cells have differentiated into matured endothelial cells as shown by the two-dimensional tube-like structures (arrow) pronounced of a lumen of a blood vessel at the arrow.
- FIG. 10B shows that there is a three-dimensional build-up of cells in multiple layers; i.e.; one on top of the other, which confirms reports that endo
- 10C shows progenitor cells growing in culture 3 weeks after plating which have the appearance of endothelial cells, and the figure confirms that the cells are endothelial cells as demonstrated by the green fluorescence of the CD34/FITC antibodies present on their surface.
- Stainless steel stents and disks are derivatized with a functional fullerene layer for attaching antibodies and/or growth factors (i.e., VEGF or Ang-2) using the following procedure:
- the surface of the SST stent or disk is activated with 0.5M HCL which also cleans the surface of any passivating contaminants.
- the metal samples are removed from the activation bath, rinsed with distilled water, dried with methanol and oven-dried at 75° C.
- the stents are then immersed in the toluene derivative solution with fullerene oxide (C 60 —O), for a period of up to 24 hours.
- the fullerene oxide binds to the stent via Fe—O, Cr—O and Ni—O found on the stent.
- the stents are removed from the derivatizing bath, rinsed with toluene, and placed in a Soxhlet Extractor for 16 hours with fresh toluene to remove any physisorbed C 60 .
- the stents are removed and oven-dried at 105° C. overnight. This reaction yields a fully derivatized stent or disk with a monolayer of fullerenes.
- step 2 a di-aldehyde molecule is formed in solution by reacting sebacic acid with thionyl chloride or sulfur oxychloride (SOCl 2 ) to form Sebacoyl chloride.
- SOCl 2 sulfur oxychloride
- the resultant Sebacoyl chloride is reacted with LiAl[t-OButyl] 3 H and diglyme to yield 1,10-decanediol as shown below:
- step 3 an N-methyl pyrolidine derivate is formed on the surface of the stent or disk (from step 1).
- the fullerene molecule is further derivatized by reacting equimolar amounts of fullerene and N-methylglycine with the 1,10-decanediol product of the reaction of step 2, in refluxing toluene solution under nitrogen for 48 hours to yield N-methyl pyrolidine-derivatized fullerene-stainless steel stent or disk as depicted below.
- the derivatized stainless steel stent or disk is washed to remove any chemical residue and used to bind the antibodies and/or (VEGF or Ang-2) using standard procedures.
- Progenitor cell are isolated from human blood as described in Example 1 and exposed to the anti-CD34 antibody coated fullerene disks. After incubation, the growth medium is removed and the samples are washed twice in PBS. Cells are fixed in 2% paraformaldehyde (PFA) for 10 minutes and washed three times, 10 minutes each wash, in PBS, to ensure all the fixing agent is removed. Each sample is incubated with blocking solution for 30 minutes at room temperature, to block all non-specific binding.
- PFA paraformaldehyde
- the samples are washed once with PBS and the exposed to 1:100 dilution of VEGFR-2 antibody and incubated overnight. The samples are subsequently washed three times with PBS to ensure all primary antibody has been removed.
- FITC-conjugated secondary antibody in blocking solution is added to each respective sample at a dilution of 1:100 and incubated for 45 minutes at room temperature on a Belly Dancer apparatus. After incubation, the samples are washed three times in PBS, once with PBS containing 0.1% Tween 20, and then again in PBS. The samples are mounted with Propidium Iodine (PI) and visualized under confocal microscopy.
- PI Propidium Iodine
- FIGS. 12A-12B are, respectively, photomicrographs of fullerene-coated control sample without antibody stained with PI ( 12 A) and anti-VEGFR-2/FITC-conjugated antibody stained.
- FIGS. 12C and 12D are photomicrographs of a sample coated with a fullerene/anti-CD34 antibody coating. As shown in the figures, the anti-CD34 antibody coated sample contains more cells attached to the surface which are VEGFR-2 positive.
- FIGS. 13A-13D are photomicrographs of cross-sections through coronary artery explants of stents which had been implanted for 4 weeks. The data show that the fullerene-coated ( FIGS. 13B and 13D ) stents inhibit excessive intimal hyperplasia at the stent site over the control (bare stent, FIGS. 13A and 13C ).
- Implantation of antibody-covered stents is performed in juvenile Yorkshire pigs weighing between 25 and 30 kg. Animal care complies with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 80-23, revised 1985). After an overnight fast, animals are sedated with ketamine hydrochloride (20 mg/kg). Following the induction of anesthesia with thiopental (12 mg/kg) the animals are intubated and connected to a ventilator that administers a mixture of oxygen and nitrous oxide (1:2 [vol/vol]). Anesthesia is maintained with 0.5-2.5 vol % isoflurane. Antibiotic prophylaxis is provided by an intramuscular injection of 1,000 mg of a mixture of procaine penicillin-G and benzathine penicillin-G (streptomycin).
- an arteriotomy of the left carotid artery is performed and a 8F-introducer sheath is placed in the left carotid artery. All animals are given 100 IU of heparin per kilogram of body weight. Additional 2,500 IU boluses of heparin are administered periodically throughout the procedure in order to maintain an activated clotting time above 300 seconds.
- a 6F guiding catheter is introduced through the carotid sheath and passed to the ostia of the coronary arteries.
- Angiography is performed after the administration of 200 ug of intra coronary nitro glycerin and images analyzed using a quantitative coronary angiography system.
- a 3F-embolectomy catheter is inserted into the proximal portion of the coronary artery and passed distal to the segment selected for stent implantation and the endothelium is denuded.
- a coated R stent incorporating an anti-CD34 antibody is inserted through the guiding catheter and deployed in the denuded segment of the coronary artery.
- Bare stainless steel stents or stents coated with the matrix but without antibodies are used as controls.
- Stents are implanted into either the Left Anterior Descending (LAD) coronary artery or the Right Coronary Artery (RCA) or the Circumflex coronary artery (Cx) at a stent to artery ration of 1.1.
- LAD Left Anterior Descending
- RCA Right Coronary Artery
- Cx Circumflex coronary artery
- the animals are sacrificed at 1, 3, 7, 14, and 28 days after stent implantation.
- the animals are first sedated and anesthetized as described above.
- the stented coronary arteries are explanted with 1 cm of non-stented vessel proximal and distal to the stent.
- the stented arteries are processed in three ways, histology, immunohistochemistry or by Scanning Electron Microscopy.
- the dissected stents are gently flushed with 10% Formalin for 30 seconds and the placed in a 10% Formalin/PBS solution until processing.
- Stents destined for immunohistochemistry are flushed with 2% Paraformaldehyde (PFA) in PBS for 30 seconds and then placed in a 2% PFA solution for 15 min, washed and stored in PBS until immunohistochemistry with rabbit anti-human VEGFR-2 or mouse anti-human Tie-2 antibodies is performed.
- PFA Paraformaldehyde
- Stents are prepared for SEM by flushing with 10% buffered Formalin for 30 seconds followed by fixation with 2% PFA with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight. Samples are then washed 3 ⁇ with cacodylate buffer and left to wash overnight. Post-fixation was completed with 1% osmium tetroxide (Sigma) in 0.1M cacodylate buffer which is followed by dehydration with ethanol (30% ethanol, 50%, 70%, 85%, 95%, 100%, 100%) and subsequent critical point drying with CO 2 . After drying, samples are gold sputtered and visualized under SEM. (Reduction in thrombotic events with heparin-coated Palmaz-Schatz stents in normal porcine coronary arteries, Circulation 93:423-430, incorporated herein by reference).
- the stented segments are flushed with 10% buffered Formalin for 30 seconds followed by fixation with 10% buffered Formalin until processed.
- Five sections are cut from each stent; 1 mm proximal to the stent, 1 mm from the proximal end of the stent, mid stent, 1 mm from the distal edge of the stent and 1 mm distal to the stent. Sections are stained with Hematoxylin & Eosin (HE) and Elastin Trichrome.
- HE Hematoxylin & Eosin
- FIGS. 14A-14G show explants taken 1 ( FIGS. 14A and 14B ) and 48 hours ( FIGS. 14C-14G ) after implantation and observed under scanning electron microscope.
- the photomicrographs clearly show that the dextran/anti-CD34 antibody-coated stents ( 14 B, 14 E-G) have capture progenitor endothelial cells as shown by the spindle-shaped appearance of the cells at higher magnification (400 ⁇ ) at 48 hours compared to the dextran-coated control ( 14 A, 14 C and 14 D).
- FIGS. 15A and 15B show, respectively, confocal photomicrographs of 48 hours explants of a dextran-plasma coated stent without antibody on is surface, and a dextran-plasma coated anti-CD34 antibody-stent of 18 mm in length.
- the stents had been implanted into the coronary artery of juvenile male Yorkshire swine.
- the explants were immunohistochemically processed and stained for VEGFR-2, followed by FITC-conjugated secondary antibody treatment and studied under confocal microscopy.
- FIGS. 15B and 15C show that the antibody containing stent is covered with endothelial cells as demonstrated by the green fluorescence of the section compared to the complete lack of endothelium on the stent without antibody ( FIG. 15A ).
- the following describes the steps for immobilizing an antibody directed toward endothelial progenitor cells cell surface antigens to a biocompatible matrix applied to an intravascular stent to which an endothelial growth factor is then absorbed for the enhanced attachment of circulating endothelial progenitor cells and their maturation to functional endothelium when in contact with blood.
- Matrix Deposition Using methods know to those skilled in the art, stainless steel stents are treated with a plasma deposition to introduce amine functionality on the stent surface. A layer of carboxy functional dextran (CMDX) will be bound to the amine functional layer deposited on the stent through the activation of the CMDX carboxyl groups using standard procedures, known as water soluble carbodiimide coupling chemistry, under aqueous conditions to which the amine groups on the plasma deposited layer to form an amide bond between the plasma layer and the functional CDMX.
- CMDX carboxy functional dextran
- Antibody Immobilization Antibodies directed toward endothelial progenitor cells cell surface antigens, e.g., murine monoclonal anti-humanCD34, will be covalently coupled with the CDMX coated stents by incubation in aqueous water soluble carbodiimide chemistry in a buffered, acidic solution.
- cell surface antigens e.g., murine monoclonal anti-humanCD34
- the device is incubated in an aqueous solution of an endothelial growth factor, e.g. Angiopoietin-2, at an appropriate concentration such that the growth factor is absorbed into the CMDX matrix.
- an endothelial growth factor e.g. Angiopoietin-2
- the treated devices are rinsed in physiologic buffered saline solution and stored in a sodium azide preservative solution.
- the above described devices when implanted in porcine coronary arteries and exposure to human blood produce an enhanced uptake and attachment of circulating endothelial progenitor cells on to the treated stent surface and accelerate their maturation into functional endothelium.
- the rapid establishment of functional endothelium is expected to decrease device thrombogenicity and modulate the extent of intimal hyperplasia.
- the following describes the steps for immobilizing an antibody directed toward endothelial progenitor cells cell surface antigens and an endothelial growth factor to a biocompatible matrix applied to an intravascular stent for the enhanced attachment of circulating endothelial progenitor cells and their maturation to functional endothelium when in contact with blood.
- Matrix Deposition Matrix Deposition: Using methods know to those skilled in the art, stainless steel stents are treated with a plasma deposition to introduce amine functionality on the stent surface. A layer of carboxy functional dextran (CMDX) is bound to the amine functional layer deposited on the stent through the activation of the CMDX carboxyl groups using standard procedures, known as water soluble carbodiimide coupling chemistry, under aqueous conditions to which the amine groups on the plasma deposited layer to form an amide bond between the plasma layer and the functional CDMX.
- CMDX carboxy functional dextran
- Antibodies directed toward endothelial progenitor cells cell surface antigens, e.g. murine monoclonal anti-human CD34, and an endothelial growth factor, e.g. Angiopoietin-2, is covalently coupled with the CDMX coated stents by incubation at equimolar concentrations in a water soluble carbodiimide solution under acidic conditions.
- the treated devices are rinsed in physiologic buffered saline solution and stored in a sodium azide preservative solution.
- the above described devices when implanted in porcine coronary arteries and exposure to human blood produce an enhanced uptake and attachment of circulating endothelial progenitor cells on to the treated stent surface and accelerate their maturation into functional endothelium.
- the rapid establishment of functional endothelium is expected to decrease device thrombogenicity and modulate the extent of intimal hyperplasia.
- Progenitor endothelial cells were isolated as described in Example 1. The cells were plated in fibronectin-coated slides and grown for 7 days in EBM-2 culture medium. Cells were fixed and stained with Propidium Iodine (PI) and a FITC-conjugated endothelial cell specific lectin. (Ulex Europaeus Uea 1) The results of these experiments are shown in FIGS. 16A and 16B . The figures show that progenitor endothelial cells are bound to the fibronectin-coated slides and that the cells express a ligand for the lectin on their surface.
- PI Propidium Iodine
- FITC-conjugated endothelial cell specific lectin Ulex Europaeus Uea 1
- Progenitor endothelial cells are transfected using electroporation of a bicistronic plasmid containing genes encoding a protein responsible for the production of adenosine and a prostate specific cell membrane protein. Both genes are under the control of their own promoter, so that the genes are expressed constitutively.
Abstract
Description
TABLE 1 | |
Endothelial cell | |
Growth Factor | specific |
Acidic fibroblast growth factor (aFGF) | No |
Basic fibroblast growth factor (bFGF) | No |
Fibroblast growth factor 3 (FGF-3) | No |
Fibroblast growth factor 4 (FGF-4) | No |
Fibroblast growth factor 5 (FGF-5) | No |
Fibroblast growth factor 6 (FGF-6) | No |
Fibroblast growth factor 7 (FGF-7) | No |
Fibroblast growth factor 8 (FGF-8) | No |
Fibroblast growth factor 9 (FGF-9) | No |
Angiogenin 1 | Yes |
Angiogenin 2 | Yes |
Hepatocyte growth factor/scatter factor (HGF/SF) | No |
Platelet-derived growth factor (PDE-CGF) | Yes |
Transforming growth factor-α (TGF-α) | No |
Transforming growth factor-β (TGF-β) | No |
Tumor necrosis factor-α (TNF-α) | No |
Vascular endothelial growth factor 121 (VEGF 121) | Yes |
Vascular endothelial growth factor 145 (VEGF 145) | Yes |
Vascular endothelial growth factor 165 (VEGF 165) | Yes |
Vascular endothelial growth factor 189 (VEGF 189) | Yes |
Vascular endothelial growth factor 206 (VEGF 206) | Yes |
Vascular endothelial growth factor B (VEGF-B) | Yes |
Vascular endothelial growth factor C (VEGF-C) | Yes |
Vascular endothelial growth factor D (VEGF-D) | Yes |
Vascular endothelial growth factor E (VEGF-E) | Yes |
Vascular endothelial growth factor F (VEGF-F) | Yes |
Placental growth factor | Yes |
Angiopoietin-1 | No |
Angiopoietin-2 | No |
Thrombospondin (TSP) | No |
Proliferin | Yes |
Ephrin-A1 (B61) | Yes |
E-selectin | Yes |
Chicken chemotactic and angiogenic factor (cCAF) | No |
Leptin | Yes |
Heparin affinity regulatory peptide (HARP) | No |
Heparin | No |
Granulocyte colony stimulating factor | No |
Insulin-like growth factor | No |
Interleukin 8 | No |
Thyroxine | No |
Sphingosine 1-phosphate | No |
Claims (16)
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